Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Imaging features of benign synovial inflammation affecting the long digital extensor tendon sheath in a dog.

A dog was evaluated for right pelvic limb lameness. Computed tomography and magnetic resonance imaging revealed an irregular, contrast-enhancing mass extending along the proximolateral right tibia, involving the long digital extensor tendon (LDET) ± cranial tibialis muscle. Pulmonary nodules, nonspecific hepatomegaly, and splenic nodules were also present. The primary differential diagnosis was soft tissue neoplasia. Surgical biopsy with histopathology revealed benign, chronic inflammation, and fibrosis. Idiopathic synovial inflammation should be included as a differential diagnosis for dogs with this combination of clinical and imaging characteristics.

Suspensory ligament size does not change after plantar fasciotomy and neurectomy of the deep branch of the lateral plantar nerve by ultrasonographic assessment.

OBJECTIVE: To determine the short-term effect of plantar fasciotomy and neurectomy (PFN) of the deep branch of the lateral plantar nerve on the proximal suspensory ligament (PSL) cross-sectional area (CSA) in horses with hindlimb proximal suspensory desmopathy (PSD). STUDY DESIGN: Analytical, observational, cohort study. SAMPLE POPULATION: Twenty-one horses. METHODS: Records of horses with chronic PSD treated by PFN were included if a preoperative ultrasonographic examination was available and at least one postoperative ultrasonographic examination. One masked observer measured the ultrasonographic cross-sectional area (CSA) of the PSL. Intraobserver reliability was determined by repeatedly measuring a subset of ultrasonographic images (n = 127). Two masked observers measured the cross-sectional area of the proximal suspensory ligament (PSL-CSA) on preoperative proton density (PD)-weighted transverse high field magnetic resonance images (n = 19 horses) . Agreements for PSL-CSA between preoperative ultrasonographic and MRI measures and between the two magnetic resonance imaging (MRI) observers were assessed. Follow up considered the horses' ability to return to exercise and their owners' satisfaction. RESULTS: The reliability of the ultrasonographic measurement of the PSL-CSA was excellent. Agreement between ultrasonographic assessment and MRI assessment of PSL-CSA was good. No difference was detected between preoperative (median, interquartile range; oblique-incidence, 2.07, 1.72-2.55; on-incidence, 2.23, 1.98-2.65) and postoperative (oblique-incidence, 2.08, 1.80-2.74; on-incidence, 2.28, 2.01-2.74) PSL-CSAs. At a median of 12 months (4-33 months), 16/20 (80%) owners reported the horse was "better" and 15/20 (75%) functioned at or above preoperative levels. CONCLUSION: Ultrasonographic measurement of the PSL-CSA was reproducible and in good agreement with MRI measurement. The PSL-CSA was not influenced by PFN. CLINICAL SIGNIFICANCE: The PSL-CSA cannot be used to guide return to function.

Detection of the Peanut Allergens Ara h 2 and Ara h 6 in Human Breast Milk: Development of 2 Sensitive and Specific Sandwich ELISA Assays.

BACKGROUND: Little is known about breast milk as a vehicle for tolerance development or sensitization to peanuts very early in life. Thus, well-characterized and highly sensitive detection systems for the reliable determination of peanut allergens in breast milk are mandatory. METHODS: For the quantification of the marker allergens Ara h 2 and Ara h 6 in the low nanogram per milliliter range in breast milk samples of a German cohort, sensitive and highly specific sandwich ELISAs were optimized and validated. RESULTS: The Ara h 2 ELISA revealed a limit of detection (LOD) of 1.3 ng Ara h 2/mL and a quantification range of 2.3-250 ng/mL, the Ara h 6 ELISA showed an LOD of 0.7 ng/mL and a working range of 1.1-14.4 ng/mL. The assays showed no relevant cross-reactivity against other potentially cross-reactive legume, seed, and tree nut extracts (<0.01%, except for Ara h 1 in the Ara h 2 ELISA <0.1%). Ara h 2 was detectable in breast milk samples from 14/40 (35%) of the participants in concentrations from 2.3 to 184 ng/mL, Ara h 6 appeared in 9/40 (22.5%) of the lactating mothers between 1.1 and 9.7 ng/mL, and 1 highly positive sample with 79 ng/mL. Both allergens appeared at the same time points, but Ara h 6 in lower concentrations than Ara h 2. CONCLUSIONS: Sensitive and specific diagnostic tools for the determination of Ara h 2 and Ara h 6 in human breast milk were established. The kinetics of secreted Ara h 2 and Ara h 6 seem to be similar but with a difference in concentration. Follow-up investigations on their tolerogenic or sensitizing properties in breast milk become now accessible.

Ex vivo diffusion tensor imaging of the plantar nerves is feasible in the horse.

OBJECTIVE: The objective of this study was to optimize an MRI-based diffusion tensor imaging (DTI) protocol for imaging the plantar nerves at the level of the tarsus in normal equine limbs. SAMPLE: 12 pelvic cadaver limbs from horses without evidence of proximal suspensory pathology were imaged with a 3T MRI system. METHODS: For diffusion-weighted imaging, b values of 600, 800, and 1,000 s/mm2 were tested. Data were processed with DSI Studio. Cross-sectional areas of the medial and lateral plantar nerve along the plantar tarsus were recorded. The length and number of fiber tracts, signal-to-noise ratio, and DTI variables were recorded. RESULTS: At the level of interest, the mean cross-sectional areas of the plantar nerves ranged from 5.03 to 7.42 mm2. The DTI maps consistently generated tracts in the region of the lateral and medial plantar nerves with DTI values in the range of values reported for peripheral nerves in humans. Our findings demonstrate that DTI of the medial and lateral plantar nerves can be performed successfully and used to generate quantitative parameters including fractional anisotropy and mean, axial, and radial diffusivity. CLINICAL RELEVANCE: Quantitative data generated with this imaging technique can be used to noninvasively characterize the microstructural integrity of neural tissue with possible applications in the evaluation of pathologic changes to the plantar tarsal and metatarsal nerves of horses with proximal suspensory desmopathy.

Influence of sedation on onset and quality of euthanasia in sheep.

The purpose of this study was to determine if dexmedetomidine administered IV prior to euthanasia in sheep affected the speed or quality of euthanasia. Twenty clinically healthy Dorset-cross adult ewes between 1 and 3years of age were enrolled in a randomized blinded experimental trial. The subjects were randomly assigned to receive dexmedetomidine 5µg/kg IV or an equivalent volume of saline. Five minutes later, euthanasia was accomplished with a pentobarbital/phenytoin overdose given IV. The time to apnea, asystole, cessation of audible heartbeat, and absence of corneal reflex were recorded by two blinded investigators. If any muscle spasms, contractions, vocalization, and/or dysrhythmias were noted, the time was recorded and type of ECG abnormality was described. An overall score of the euthanasia event was assigned using a numeric rating scale (NRS) after the animal was declared dead. The time to loss of corneal reflex was significantly longer in sheep given dexmedetomidine compared with those who received saline (P=0.03). Although vocalization was observed only in some animals premedicated with dexmedetomidine, no significance was found for this event and no other significant differences between groups were noted. Dexmedetomidine at 5µg/kg IV 5min prior to injection of pentobarbital/phenytoin for euthanasia did not substantially affect the progress of euthanasia. Dexmedetomidine may be given to sedate sheep prior to euthanasia without concern for it adversely affecting the progress of euthanasia, however vocalization may occur.

MRI-Based Assessment of Intralesional Delivery of Bone Marrow-Derived Mesenchymal Stem Cells in a Model of Equine Tendonitis.

Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 106 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migration, or trilineage differentiation due to the presence of SPIOs. Limbs were imaged on a 1.5-tesla clinical MRI scanner postmortem before and after injection to determine the extent of tendonitis and detect SPIO MSCs. Clusters of labeled cells were visible as signal voids in 6/6 subjects. Coalescing regions of signal void were diffusely present in the peritendinous tissues. Although previous reports have determined that local injury retains cells within a small radius of the site of injection, our study shows greater than expected delocalization and relatively few cells retained within collagenous tendon compared to surrounding fascia. Further work is needed if this is a reality in vivo and to determine if directed intralesional delivery of MSCs is as critical as presently thought.

Determination of the minimum alveolar concentration of isoflurane that blunts adrenergic responses in sheep and evaluation of the effects of fentanyl.

OBJECTIVE: To determine the minimum alveolar concentration that blunts adrenergic responses (MACBAR) for isoflurane and evaluate effects of fentanyl on isoflurane MACBAR in sheep. ANIMALS 13 healthy adult Dorset-cross adult ewes. PROCEDURES: In a crossover design, each ewe was anesthetized 2 times for determination of isoflurane MACBAR. Anesthesia was induced with propofol administered IV. Sheep initially received fentanyl (5 µg/kg, IV, followed by a constant rate infusion of 5 µg/kg/h) or an equivalent volume of saline (0.9% NaCl) solution (control treatment). After a washout period of at least 8 days, the other treatment was administered. For MACBAR determination, a mechanical nociceptive stimulus (ie, sponge forceps) was applied at the coronary band for 1 minute. The MACBAR values of the 2 treatments were compared by means of a paired t test. During MACBAR determination, blood samples were collected for measurement of plasma fentanyl concentration. RESULTS: Mean ± SD isoflurane MACBAR of the fentanyl and control treatments was 1.70 ± 0.28% and 1.79 ± 0.35%, respectively; no significant difference was found between the 2 treatments. Plasma concentration of fentanyl reached a median steady-state concentration of 1.69 ng/mL (interquartile range [25th to 75th percentile], 1.47 to 1.79 ng/mL), which was maintained throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of fentanyl at 5 µg/kg, IV, followed by a constant rate infusion of the drug at 5 µg/kg/h did not decrease isoflurane MACBAR. Further studies to determine the effect of higher doses of fentanyl on inhalation anesthetic agents and their potential adverse effects are warranted.

Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury.

The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration.

Performance of ELISA and PCR methods for the determination of allergens in food: an evaluation of six years of proficiency testing for soy (Glycine max L.) and wheat gluten (Triticum aestivum L.).

For the routine detection of allergens in foods, PCR and/or ELISA methods are employed. To assess the suitability of these methods, proficiency tests (PTs) could be used as a valuable instrument. It is a common practice to evaluate the results with respect to the experimentally obtained robust mean without considering the actual allergen content. In the present study, an overview is given of the results of allergen PTs for the determination of soy and gluten conducted by Dienstleistung Lebensmittel Analytik GbR (DLA). A total of 16 PTs were evaluated with respect to the comparison of PCR and ELISA performances and a new focus on the actually spiked values. The analytes were added in the ranges of 7.8-6264 mg/kg (gluten) and 184-5500 mg/kg (soy protein) in differently composed matrices such as pastry, infant food, and sausage meat. The evaluation of the PTs showed a widely reliable qualitative detection of both allergens by PCR methods. ELISA performances differed for soy and gluten. Although a high number of false-negative results occurred for the detection of soy, the qualitative detection of gluten was appropriate. Quantitative results showed obvious test kit-specific differences for the ELISA methods, but the limits of quantification were suitable for gluten determination. Both ELISA and PCR methods demonstrated their valuable contribution in food allergen determination.